EZH2 polyclonal antibody - Classic

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Figure 1. ChIP results obtained with the Diagenode antibody directed against EZH2ChIP assays were performed using K562 cells, the Diagenode antibody against EZH2 (Cat. No. C15410039) and optimized PCR primer sets for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010055), using sheared chromatin from 4 million cells. A titration of the antibody consisting of 1, 2. 5 and 10 µg per ChIP experiment was analysed. IgG (2 µg/IP) was used as negative IP control. Quantitative PCR was performed with primers for MYT1 and HOXA9, used as positive control targets, and for the coding regions of the active CCT5 and EIF2S3 genes, used as negative controls. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

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Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against EZH2 ChIP was performed on sheared chromatin from 4 million K562 cells using 2 µg of the Diagenode antibody against EZH2 (Cat. No. C15410039) as described above. The IP’d DNA was subsequently analysed on an Illumina HiSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 51 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the peak distribution along the short arm and a 6 Mb region containing several enriched regions of human chromosome 3 (figure 2A and B, respectively), and in two genomic regions containing the MYT1 gene on chromosome 20 and the HOX cluster on chromosome 7 (figure 2C and D).

Figure 3. Western blot analysis using the Diagenode antibody directed against EZH2 Nuclear extracts of HeLa cells (40 µg) were analysed by Western blot using the Diagenode antibody against EZH2 (Cat. No. C15410039) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest (expected size 85 kDa) is indicated on the right; the marker (in kDa) is shown on the left.

Figure 4. Western blot analysis using the Diagenode antibody directed against EZH2 Whole cell extracts (40 µg) from HeLa cells transfected with EZH2 siRNA (lane 2) and from an untransfected control (lane 1) were analysed by Western blot using the Diagenode antibody against EZH2 (Cat. No. C15410039) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.

Figure 5. Immunofluorescence using the Diagenode antibody directed against EZH2 HeLa cells were stained with the Diagenode antibody against EZH2 (cat. No. C15410039) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 1% BSA. The cells were immunofluorescently labelled with the EZH2 antibody (left) diluted 1:1,000 in blocking solution followed by an anti-mouse antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.

EZH2 (UniProt/Swiss-Prot entry Q15910) is a histone-lysine methyltransferase which methylates ‘Lys-9’ and ‘Lys-27’ of histone H3, leading to transcriptional repression. It is a member of the polycomb group (PcG) family which form multimeric protein complexes and are involved in maintaining the transcriptional repressive state of genes over successive cell generations. The EZH2 activity is dependent on the association with other components of the PRC2 complex (EED, EZH2, SUZ12/JJAZ1, RBBP4 and RBBP7). EZH2 may play a role in the hematopoietic and central nervous systems. Over-expression of EZH2 is observed during advanced stages of prostate cancer and breast cancer.

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A new metabolic gene signature in prostate cancer regulated by JMJD3 and EZH2.Daures M, Idrissou M, Judes G, Rifaï K, Penault-Llorca F, Bignon YJ, Guy L, Bernard-Gallon DHistone methylation is essential for gene expression control. Trimethylated lysine 27 of histone 3 (H3K27me3) is controlled by the balance between the activities of JMJD3 demethylase and EZH2 methyltransferase. This epigenetic mark has been shown to be deregulated in prostate cancer, and evidence shows H3K27me3 enri...

STAT5BN642H is a driver mutation for T cell neoplasiaPham H.T.T. et al.STAT5B is often mutated in hematopoietic malignancies. The most frequent STAT5B mutation, Asp642His (N642H), has been found in over 90 leukemia and lymphoma patients. Here, we used the Vav1 promoter to generate transgenic mouse models that expressed either human STAT5B or STAT5BN642H in the hematopoietic compartment...

H19 lncRNA controls gene expression of the Imprinted Gene Network by recruiting MBD1.Monnier P, Martinet C, Pontis J, Stancheva I, Ait-Si-Ali S, Dandolo LThe H19 gene controls the expression of several genes within the Imprinted Gene Network (IGN), involved in growth control of the embryo. However, the underlying mechanisms of this control remain elusive. Here, we identified the methyl-CpG-binding domain protein 1 MBD1 as a physical and functional partner of the H19 ...

Silencing of Kruppel-like factor 2 by the histone methyltransferase EZH2 in human cancer.Taniguchi H, Jacinto FV, Villanueva A, Fernandez AF, Yamamoto H, Carmona FJ, Puertas S, Marquez VE, Shinomura Y, Imai K, Esteller MThe Kruppel-like factor (KLF) proteins are multitasked transcriptional regulators with an expanding tumor suppressor function. KLF2 is one of the prominent members of the family because of its diminished expression in malignancies and its growth-inhibitory, pro-apoptotic and anti-angiogenic roles. In this study, we ...

Enhancer of Zeste 2 (EZH2) is up-regulated in malignant gliomas and in glioma stem-like cells.Orzan F, Pellegatta S, Poliani PL, Pisati F, Caldera V, Menghi F, Kapetis D, Marras C, Schiffer D, Finocchiaro GAIMS: Proteins of the Polycomb repressive complex 2 (PRC2) are epigenetic gene silencers and are involved in tumour development. Their oncogenic function might be associated with their role in stem cell maintenance. The histone methyltransferase Enhancer of Zeste 2 (EZH2) is a key member of PRC2 function: we have in...

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